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The effects of various lesions and knife-cuts on septal and amygdala kindling in the rat
Brain Research, 454 (1988) 264-274 Elsevier
264 BRE 13729
The effects of various lesions and knife-cuts on septal and amygdala kindling in the rat R.J. Racine 1, G. Paxinos 2, J.M.
M o s h e r 1 a n d E . W . Kairiss 1
1Department of Psychology, McMaster University, Hamilton, Ont. (Canada) and 2School of Psychology, University of New South Wales, Kensington, N.S. W. (Australia) (Accepted 28 January 1988)
Key words: Epilepsy; Kindling; Amygdala; Pyriform cortex; Septal area; Hippocampus; Lesion
Large bilateral aspiration lesions of the hippocampus had no significant effect on septal kindling, whereas large bilateral DC lesions of the pyriform lobe resulted in a small but significant increase in the number of septal stimulations required to complete kindling. Bilateral aspiration lesions of the dorsal hippocampus or large bilateral DC lesions of the ventral hippocampus had no effect on amygdala kindling. Small DC lesions of the stria terminalis significantly facilitated amygdala kindling. Unilateral or bilateral ventral knife-cuts delivered in a coronal plane anterior to the amygdala, disrupting communication with anterior pyriform structures, produced a small but nearly significant increase in the number of stimulations required for amygdala kindling. Similar cuts placed posterior to the amygdala, disrupting communication with the hippocampus, significantly facilitated kindling. Cuts that were medially placed, to disrupt the ventral amygdala-fugal pathway, had no effect on amygdala kindling. These results show that the hippocampus is not critical for either septal or amygdala kindling. The pyriform lobe structures appear to play a facilitatory role in kindling, but none of the lesions or knifecuts were capable of blocking or even severely retarding kindling. INTRODUCTION
In the previous p a p e r 23, we r e p o r t e d that the pyriform cortex was the most reactive region in the rat brain for the d e v e l o p m e n t of inter-ictal spikes (IIS) in the kindled p r e p a r a t i o n . A s kindling progressed, however, it a p p e a r e d that other structures could also function as IIS generators. W e suggested that the pyriform cortex played an i m p o r t a n t role in the early stages of kindling, regardless of which structure received the kindling stimulation. It remains to be seen whether kindling would be disrupted in the absence of an intact pyriform lobe. T h e r e is also controversy about the role of the h i p p o c a m p u s in kindling. Several authors have suggested that activation of the hippocampus is a m a j o r stage in the d e v e l o p m e n t of kindled seizures 4'8'15'17'26'3°. W e have not found the hippocampus to be particularly responsive, however, with respect to the generation of IIS. The first e x p e r i m e n t r e p o r t e d below c o m p a r e d the
effects of bilateral pyriform lobe lesions with bilateral h i p p o c a m p a l lesions on kindling in the lateral septal nucleus. The second e x p e r i m e n t c o m p a r e d the effects of dorsal and ventral h i p p o c a m p a l lesions on amygdala kindling. This comparison was m a d e because our previous experiments indicated that the ventral h i p p o c a m p u s was m o r e reactive than the dorsal hippocampus in the generation of IIS 9'23. Finally, we tested the effects of knife cuts that were a i m e d at disrupting, in different groups, the communications between the amygdala and its various target sites, including the anterior pyriform cortex and the hippocampus. The ,results support the hypothesis that the pyriform cortex plays a facilitatory role in the early stages of kindling. On the o t h e r hand, it does not a p p e a r to be necessary for kindling. The hypothesis that the hippocampus plays a critical role in either the p r o p a gation or the d e v e l o p m e n t of epileptiform activity was not s u p p o r t e d by the results of the following experiments.
Correspondence: R.J. Racine, Department of Psychology, McMaster University, Hamilton, Ont. L8S 4K1, Canada. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
265 MATERIALSAND METHODS
Experiment 1 - - the effect of pyriform lobe vs hippocampal lesions on septal kindling All hooded rats were purchased from Charles River Breeding Farms and weighed 275-325 g at the time of surgery. Pyriform lobe lesions. Several attempts were made to produce large bilateral lesions in the amygdala/pyriform area before a final technique was selected. Radio frequency lesions of the pyriform lobe were found to be unreliable. Although we knew from pilot experiments that small electrolytic lesions in the pyriform region could be epileptogenic, and facilitate kindling (see also refs. 2, 3 and 16), we had found that large electrolytic lesions were less epileptogenic (possibly because the intact tissue was further removed from the site of ion deposits). Consequently, we selected this technique for producing large bilateral lesions in the pyriform lobe. The possibility of lesion-induced activity at distant sites remained, however, so we included those animals that had sustained sufficiently large radio frequency lesions. Three Teflon-coated stainless steel electrodes 240 ~zm in diameter, were implanted along the anterior/ posterior axis of the pyriform lobe in 20 animals. A bipolar stimulating electrode was also implanted into the lateral septal nucleus. At the time of surgery, a 2 mA anodal current was passed via the anterior electrodes, 30 s to one hemisphere followed by 30 s to the other. One week later, similar currents were passed via the middle 2 electrodes. The final 2 lesions were made one week later. It was necessary to produce these lesions in stages to ensure the survival of the animal. After a final one week recovery period, kindling was begun. Hippocampal lesions. An oval shaped opening was cut into the skull overlying the hippocampus in 8 animals. The overlying neocortex and the dorsal hippocampus were then removed by aspiration. A Tefloncoated stainless-steel bipolar stimulating electrode was implanted into the lateral septal nucleus. Two weeks following surgery, kindling was begun. Control animals and kindling procedure. Ten control animals were prepared with stimulating electrodes in the lateral septal nucleus. Kindling was begun 2-4 weeks following surgery. All animals were stimulated once per day with a 1-s train of biphasic,
rectangular pulses at 60 Hz. The current was initially set at 400/~A (800 ~A peak to peak). If this intensity did not produce an epileptiform afterdischarge (AD), then it was raised in 200-#A steps until an AD was produced. Similar intensity increases were made in randomly selected control animals. It was our intention to keep the current intensity well above threshold. We have found in previous experiments that kindling rates are highly variable when the stimulation intensity is near threshold levels (see also ref. 21). Stimulation was continued until the animal showed 2 consecutive generalized convulsions, consisting of a bilateral forelimb clonus, clonic rearing and a loss of postural control (falling). At the end of this, and all subsequent experiments, the brains were removed, fixed, sectioned and stained with thionin. Electrode placements and the extent of lesions (or knife-cuts) were then determined.
Experiment 2 - - the effect of dorsal hippocampal vs ventral hippocampal/entorhinal lesions on amygdala kindling The dorsal lesions were produced as in the previous experiments. Radio frequency lesions were attempted for the ventral lesions but were again found to be unreliable. Consequently, the ventral lesions were made electrolytically. In order to ensure that these lesions included ventral hippocampus and adjacent entorhinal cortex, the electrode was lowered, at an angle of 30° anterior to posterior, until the 1.0-mm bared tip was in the mid-caudal portion of the entorhinal cortex. A 2-mA current was then passed via the electrode for 30 s, after which the electrode was retracted dorsorostrally until the tip was presumed to be in the ventral hippocampus. The 2-mA current was reapplied. The electrode was moved to the other hemisphere and the procedure repeated. A bipolar stimulating electrode was then placed in the basolateral nucleus of the amygdala. A control group of 14 animals were prepared with a bipolar stimulating electrode in the amygdala. Two to 4 weeks following surgery, kindling was begun. The kindling procedure was as described above.
Experiment 3 - - the effect of knife-cuts in various amygdalar pathways Ninety-eight male hooded rats were used in this
266 experiment. Knife-cuts were made using the encannulated knife technique of Sclafani and Grossman 27. In this procedure, a cannula with an angled tip is lowered to a point above the intended site of transection. The wire is then pushed out of the cannula for several millimeters (depending upon the intended width of the cut). The cannula is lowered for the intended depth of the cut, after which the wire and the cannula are withdrawn. Knife cuts were made in all pathways except the stria terminalis, where we were unable to make reliable transections using this technique (the compact fiber bundle may have been deflected rather than cut by the curved wire knife). Consequently, we used electrolytic lesions to damage this pathway. Anterior coronal cuts. One group of 10 animals received unilateral knife cuts in a ventral coronal plane anterior to the amygdala. These cuts were intended to disrupt communication anteriorly with the pyriform cortex, as well as with medial structures (via amygdalar fibers that run anteriorly before turning mediaUy11). An additional group of 10 animals re-
ceived bilateral cuts in the same region. Medial cuts. Cuts were positioned medial to the amygdala, to disrupt ventral-amygdalofugal (VAF) fibers, in 20 animals. Ten of these received unilateral cuts, while the remaining 10 received bilateral cuts. Posterior coronal cuts. Cuts were made in a ventral coronal plane posterior to the amygdala in 20 animals (10 unilateral and 10 bilateral). These cuts were intended to disrupt communication with the posterior entorhinal cortex and the hippocampus 12. Stria terminalis lesions. Electrodes made of Tefloncoated 240/~m nicrome wire were used to pass 1 mA of anodal current in the region of the stria terminalis for 20 s. Ten animals received unilateral lesions and 10 animals received bilateral lesions. Control groups. The encannulated knife was lowered, without extending the knife, in a group of 18 control rats. Six groups of 3 control rats received the cannulae in the same placements as in the 6 knife cut groups. Kindling procedures. After the cuts were made, all groups received a bipolar nichrome wire electrode into both the amygdala and the dorsal hippocampus. The kindling stimulation was applied to the amygdala, and kindling procedures were as previously described. RESULTS
/
Fig. 1. For all experiments reported in this paper, electrode tips were found to be located within the regions indicated. (Atlas diagrams are from Paxinos and Watson19.)
Experiment 1 - - the effect of pyriform lobe vs hippocampal lesions on septal kindling The location of electrode tips for this and subsequent experiments is shown in Fig. 1. The extent of the lesions for this experiment is shown in Fig. 2. Two of the animals with hippocampal lesions died during the course of kindling. It was difficult to produce large bilateral lesions in the pyriform lobe, even in 3 stages, without killing the animals. Consequently, from the 20 animals that sustained lesions, we obtained only 10 survivors. Several of these had to be force fed during the first week after surgery. Feeding behavior eventually recovered, however, and the surviving animals, although lighter than controls, appeared active and healthy. As can be seen in Fig. 2, the resulting pyriform lobe lesions were not complete but the total damage was comparable to that of the hippocampal group. The electrode of one of the control animals was outside of the amygdala, so this ani-
267
PYRIFORM LOBE LESION SMALLEST
LARGEST
1.7MM
"0.8MM
-3.3MM
DORSAL HPC LESION SMALLEST
LARGEST
-3.3MN
"4.3MM
-5.3MM
Fig. 2. The smallest and largest pyriform lobe lesions as well as the smallest and largest hippocampal lesions are shown above. Note that the pyriform lobe lesions are represented by cross-section through the c e n t e r s of the 3 anterior/posterior lesions placed in those lobes. There was considerable reduction in volume of damage half way between lesion sites (about 70-100% reduction for the small lesions and a 30-60% reduction for the large lesions).
268 mal's data were excluded from the analysis. The data and analyses are presented in Table I. As can be seen, the group with the pyriform lesion required a significantly greater number of stimulations to complete kindling than either the control group or the group with the hippocampal lesions. The mean kindling rate was actually somewhat faster in the group with hippocampal lesions, but the differences did not reach significance. None of the measures of A D strength (duration, spike amplitude, etc.) were significantly different between the groups, although the A D durations in the group with pyriform lobe lesions were somewhat shorter than controls upon completion of kindling (P = 0.1). None of the animals with pyriform lesions required increases in the intensity of stimulation in order to evoke ADs. Three of the animals with hippocampal lesions required a 100% increase in stimulation intensity. Experiment 2 - - the effects o f dorsal hippocampal vs ventral hippocampal/entorhinal lesions on amygdala kindling The extent of the dorsal and ventral lesions are shown in Fig. 3. Two animals with dorsal lesions were lost during the course of kindling (they had received only 2 and 4 stimulations, respectively). The remaining animals were healthy and active. Table II shows the data and analyses. There were no significant differences between the groups in the number of stimuTABLE I Septal kindling Number of ADs to first stage 5 convulsion. Lesion Amygdala/pyriform (n=lO)
Hippocampus (n=6)
Control (n = 9)
41.6 (26-59)
23.5 (10-45)
30.3 (13-49)
Experiment 3 - - the effects o f knife cuts in various amygdalar pathways Minimal, maximal and/or representative cuts (or lesions) for each structure are shown in Fig. 4. Data and analyses are shown in Table III. There were no significant differences between the effects of unilateral and bilateral cuts, so the groups were pooled for pairwise comparisons. Stria terminalis lesions produced a significant facilitation of kindling. Cuts in the coronal plane between the amygdala and the ventral hippocampus also resulted in a significant facilitation of kindling. Cuts in the coronal plane anterior to the amygdala retarded kindling, but the effect was not very large. Although there were no significant differences in A D durations at any time during the experiment, there appeared to be a tendency towards longer first trial A D durations in the groups with stria terminalis lesions or posterior coronal cuts. Propagation to the hippocampus, however, was strikingly affected in the groups with posterior ventral coronal cuts. These animals showed no sign of propagated activity to the hippocampus during early stages of kindling and little or no propagation even after generalized convulsions had developed. If propagated activity was measured in terms of either absolute spike amplitude or amplitude relative to background E E G amplitude (signal to noise) there was no overlap between the posterior ventral coronal cut groups and any other group tested. Examples of these discharges are shown in Fig. 5. TABLE II
ANOVA Between Within Total
lations required to reach a stage 5 convulsion. There were also no significant differences in any of the measures of the A D , except that all animals with ventral hippocampal lesions required a 100-200% increase in stimulation intensity in order to trigger ADs.
Amygdala kindling Number of ADs to first stage 5 convulsion.
ss df 1344.1 2 3015.9 22 4360.0 24
Pairwise Pyriform vs control t = 2.25, P = 0.036
MS 672.1 137.1
F 4.9
Pyr vs hpc t = 3.02, P = 0.009
P 0.017
Control vs hpc t = 1.01, N.S.
Lesion Dorsal hippoeampus n=6
Ventralhippocampal
Control
.13.3 (8-27) ANOVA N.S.
12.4 (10-18) N.S.
12.1 (8-23) N.S.
269
DORSAL HPC LESION SMALLEST
LARGEST
-3,
-4
-5,
VENTRAL HPC LESION SMALLEST
LARGEST
Fig. 3. The smallest and largest lesions of the dorsal hippocampus and ventral hippocampus/entorhinal cortex are shown above.
270 A further difference between the groups with either posterior ventral coronal cuts or stria terminalis lesions and the other groups was that post-ictal spiking appeared
earlier in the
former
groups
clear that the hippocampus is not critical for either septal Or amygdala kindling. Large dorsal hippocampal lesions affected neither septal nor amygdala kindling. Large ventral hippocampal/entorhinal le-
DISCUSSION
sions had no effect on amygdala kindling. Although epileptogenic effects of the D C lesions on intact pyriform lobe tissue might have masked an otherwise de-
There are several conclusions that can be drawn on the basis of these lesion and knife-cut data. First, it is
pressant effect of the lesions, Expt. 3 showed that a blockade of c o m m u n i c a t i o n between the amygdala and the hippocampus (confirmed by recording hippo-
(Fig. 5).
A
B
C
STRIA LESION
.
V A F KNIFE CUT
A
B
A
B
.0
POSTERIOR CORONAL CUT
ANTERIOR CORONAL CUT
Fig. 4. The extent of the lesions or knife cuts are illustrated above. The smallest (A), most representative (B) and largest (C) lesions of the stria terminalis are shown in the top row. The number in the upper left hand corner indicates the AP coordinate with reference to bregma (Pellegrino and Cushman2°). A typical ventral-amygdalofugal (VAF) cut is shown in the second row. The extent of the cut is indicated by the vertical line in each section. There was little variation in these cuts and nearly all reached the base of the brain between AP coordinates +2.0 and -0.4 ram. The posterior and anterior coronal cuts are illustrated in the bottom row. The largest (A) and smallest (B) cuts, as reconstructed from sagital sections, are shown for both types of cuts.
271 TABLE III Number of afterdischarges (ADs) required to kindle to generalized seizure Unilateral cuts
Bilateral cuts
Stria terminalis
r X
(5-12) 7.8 n=9
(4-9) 6.4 n=8
Posterior coronal
r X
(4-12) 7.9 n=9
(5-11) 7.8 n=9
VAF
r X
(6-14) 10.8 n = 10
(7-31) 12.9 n = 10
Anterior coronal
r X
(10-21) 13.7 n=10
(9-19) 14.2 n=9
Control
r X
(8-13) 10.8 n=8
(8-15) 11.3 n=8
ANOVA Source Total Placement Uni- or bilateral Placement unior bilateral Error
SS 11326 593.7
df 89 4
ms 148.4
2.3
1
2.3
29.5 887
4 80
7.4 11.1
Pairwise (Tukey): Stria vs control Posterior vs control VAF vs control Anterior vs control
F 13.4
-
P 0.001
0.21
N.S.
0.66
N.S. -
P < 0.01 P < 0.05 N.S. P < 0.09
campal activity) was actually a c c o m p a n i e d by a facilitation of kindling. These results are different from those of Savage et al. 26 who r e p o r t e d that knife cuts in the entorhinal cortex r e t a r d e d amygdala kindling, and of Dashieff and M c N a m a r a 4 who r e p o r t e d that colchicine application to the d e n t a t e gyrus r e t a r d e d entorhinal cortex kindling. T h e r e are several possible explanations for these differences. Most of the experiments that have tested the effects of lesions have used kindling stimulations that were below or just above threshold intensity (for A D ) . Pinel et al. 21 have shown that kindling rates can be quite variable near threshold, and are usually prolonged. This appears to be due to the fact that n e a r threshold stimulation often produces weak discharges that contribute little to the kindling process 21 (Racine, unpub-
lished observations). Even small alterations in A D threshold might account for an a p p a r e n t increase in kindling rates when the stimulation intensity is held near the threshold levels. W e have used stimulation levels that were clearly suprathreshold to ensure that we were measuring kindling rates and not a secondary effect of threshold change. The colchicine results of Dashieff and M c N a m a r a 4 could be due to an effect of the drug outside of the site of application. The authors state that they had to exclude 9 animals that showed clear entorhinal cortex damage. M a n y of their remaining animals showed substantial increases in threshold and reduction of A D durations. Nevertheless, they still required fewer total seconds of A D (though an increased n u m b e r of A D s ) to complete kindling. The authors conceded that the colchicine may have had some effect outside the injected area. Also, Sutula et al. 28 found no effect of colchicine, administered to the dentate, on entorhinal cortex kindling (although they did find an increase in A D threshold). T h e r e is certainly no question, from our results, that the h i p p o c a m p u s is n o t a critical link in the d e v e l o p m e n t and p r o p a g a t i o n of kindled seizures, and its activation m a y even slow down the development of kindling. This interpretation is consistent with the fact that the h i p p o c a m p u s is one of the slowest kindling sites in the brain 1°'22'24. In fact, when convulsions do finally a p p e a r during h i p p o c a m p a l kindling, they often occur during a p e r i o d of s u p p r e s s e d discharge in the h i p p o c a m p u s 22. This p e r i o d of suppression follows the h i p p o c a m p a l A D , and the epileptiform activity of other limbic sites is often of largest amplitude during this period. A l t h o u g h the ventral h i p p o c a m p a l / e n t o r h i n a l lesions did p r o d u c e large increases in A D thresholds, this m a y have been due to an e n c r o a c h m e n t of the lesion into the amygdala (near the vicinity of the stimulating electrode). W e had earlier a t t e m p t e d to test amygdala kindling in animals with pyriform lobe lesions anterior to the electrode. W e could not even elicit A D s from these animals, p r e s u m a b l y for the same reason. A n o t h e r conclusion that can be drawn is that the pyriform lobe region appears to be facilitatory for kindling. Medium-sized bilateral lesions of the pyriform lobe significantly r e t a r d e d septal kindling, and ventral coronal knife-cuts b e t w e e n the a m y g d a l a and the anterior pyriform cortex r e t a r d e d a m y g d a l a
272 ANTERIOR VENTRAL CORONAL CUT S No. 4
v
16
:b,W,~vb.:::-:L.W~t.:.. . . . . . .
POSTERIOR VENTRAL CORONAL CUT S No. $ I
]
7
:'/q~ ;
II~.~V
......
Fig. 5. The development of the afterdischarge (AD) during kindling is shown for 2 representative animals. The top 4 traces were taken from an animal with an anterior ventral coronal cut. The 1st, 5th and 16th ADs are shown. The last AD was accompanied by a fully generalized convulsion. The development in this animal was typical for that group and for every other group except those with posterior ventral coronal cuts. Traces from a typical animal from the latter group are shown in the bottom 4 traces. The 1st, 3rd and 7th ADs are shown for this animal. The 7th AD was accompanied by a fully generalized convulsion. Note that the propagation to the hippocampus (HPC) is strong from day 1 in the animal with the anterior cut and never develops for the animal with the posterior cut. Also, note the post-ictal spikes that appear near the end of the last trace in the animal with the posterior cuts. These post-ictal spikes developed within the first few stimulations in the posterior cut animals, but they required at least 2 or 3 additional ADs for animals in the other groups.
kindling. The results of both the hippocampal lesions and the pyriform lobe lesions are consistent with those presented in the previous paper in which we reported that inter-ictal spikes appear to develop more readily in the pyriform region than in the hippocampus 23. As mentioned in the Methods section, we had initially attempted to produce these lesions with the radio frequency technique. Although this technique proved unreliable in our hands, we did eventually obtain 3 animals with lesions as large as those produced here. Those animals also required a greater than normal number of stimulations to kindle. These results are consistent with those of Le Gal La Salle 13who re-
ported that amygdala lesions retarded kindling from the bed nucleus of the stria terminalis and of Cain 1 who found that amygdala lesions retarded olfactory bulb kindling. The stria terminalis lesions facilitated amygdala kindling. This finding confirms that reported by Engel and Katzman 7 who also found a significant facilitatory effect of stria terminalis lesions. They suggested that this effect was due to a disruption of ascending noradrenergic fibers that run through the stria terminalis on their way to the amygdala 14,29. Another possibility is that the amygdalar projections, via the stria terminalis, to diencephalic structures are predominantly inhibitorys,6,25. Disruption of these fibers
273 might then be expected to facilitate the relay of epileptogenic activity into diencephalic and brainstem structures over remaining pathways. These alternative possibilities also apply to the facilitation pro-
the nucleus accumbens, ventral pallidum and substantia i n n o m i n a t a may be important in the propagation of the discharge from pyriform lobe sites (see
duced by posterior ventral coronal cuts. Either of
also ref. 18). We have attempted several times to place lesions in these structures, but the survival rate
these pathways could be inhibitory in either direction resulting in a disinhibition of the primary (stimu-
has been near zero. A final conclusion to be drawn from these results is
lated) site or of one or more secondary sites to which the primary site projects. The ventral amygdalofugal cuts had no significant
that it is very difficult to block kindling. None of the lesions or knife-cuts in these experiments did so. Even the pyriform lobe lesions only produced a rela-
effect. This was surprising for us because we had
tively mild suppression of kindling. This provides ad-
found what appeared to be a retarding effect in a pilot experiment prior to beginning this study. Those
ditional support for our argument in the previous paper 23 that m a n y sites are capable of developing a
cuts were more anterior to the 'corrected' placements used here. If the pilot results reflected a genuine effect, there is a possibility that the region of
chronic epileptogenic responsivity, with the pyriform
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264 BRE 13729
The effects of various lesions and knife-cuts on septal and amygdala kindling in the rat R.J. Racine 1, G. Paxinos 2, J.M.
M o s h e r 1 a n d E . W . Kairiss 1
1Department of Psychology, McMaster University, Hamilton, Ont. (Canada) and 2School of Psychology, University of New South Wales, Kensington, N.S. W. (Australia) (Accepted 28 January 1988)
Key words: Epilepsy; Kindling; Amygdala; Pyriform cortex; Septal area; Hippocampus; Lesion
Large bilateral aspiration lesions of the hippocampus had no significant effect on septal kindling, whereas large bilateral DC lesions of the pyriform lobe resulted in a small but significant increase in the number of septal stimulations required to complete kindling. Bilateral aspiration lesions of the dorsal hippocampus or large bilateral DC lesions of the ventral hippocampus had no effect on amygdala kindling. Small DC lesions of the stria terminalis significantly facilitated amygdala kindling. Unilateral or bilateral ventral knife-cuts delivered in a coronal plane anterior to the amygdala, disrupting communication with anterior pyriform structures, produced a small but nearly significant increase in the number of stimulations required for amygdala kindling. Similar cuts placed posterior to the amygdala, disrupting communication with the hippocampus, significantly facilitated kindling. Cuts that were medially placed, to disrupt the ventral amygdala-fugal pathway, had no effect on amygdala kindling. These results show that the hippocampus is not critical for either septal or amygdala kindling. The pyriform lobe structures appear to play a facilitatory role in kindling, but none of the lesions or knifecuts were capable of blocking or even severely retarding kindling. INTRODUCTION
In the previous p a p e r 23, we r e p o r t e d that the pyriform cortex was the most reactive region in the rat brain for the d e v e l o p m e n t of inter-ictal spikes (IIS) in the kindled p r e p a r a t i o n . A s kindling progressed, however, it a p p e a r e d that other structures could also function as IIS generators. W e suggested that the pyriform cortex played an i m p o r t a n t role in the early stages of kindling, regardless of which structure received the kindling stimulation. It remains to be seen whether kindling would be disrupted in the absence of an intact pyriform lobe. T h e r e is also controversy about the role of the h i p p o c a m p u s in kindling. Several authors have suggested that activation of the hippocampus is a m a j o r stage in the d e v e l o p m e n t of kindled seizures 4'8'15'17'26'3°. W e have not found the hippocampus to be particularly responsive, however, with respect to the generation of IIS. The first e x p e r i m e n t r e p o r t e d below c o m p a r e d the
effects of bilateral pyriform lobe lesions with bilateral h i p p o c a m p a l lesions on kindling in the lateral septal nucleus. The second e x p e r i m e n t c o m p a r e d the effects of dorsal and ventral h i p p o c a m p a l lesions on amygdala kindling. This comparison was m a d e because our previous experiments indicated that the ventral h i p p o c a m p u s was m o r e reactive than the dorsal hippocampus in the generation of IIS 9'23. Finally, we tested the effects of knife cuts that were a i m e d at disrupting, in different groups, the communications between the amygdala and its various target sites, including the anterior pyriform cortex and the hippocampus. The ,results support the hypothesis that the pyriform cortex plays a facilitatory role in the early stages of kindling. On the o t h e r hand, it does not a p p e a r to be necessary for kindling. The hypothesis that the hippocampus plays a critical role in either the p r o p a gation or the d e v e l o p m e n t of epileptiform activity was not s u p p o r t e d by the results of the following experiments.
Correspondence: R.J. Racine, Department of Psychology, McMaster University, Hamilton, Ont. L8S 4K1, Canada. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
265 MATERIALSAND METHODS
Experiment 1 - - the effect of pyriform lobe vs hippocampal lesions on septal kindling All hooded rats were purchased from Charles River Breeding Farms and weighed 275-325 g at the time of surgery. Pyriform lobe lesions. Several attempts were made to produce large bilateral lesions in the amygdala/pyriform area before a final technique was selected. Radio frequency lesions of the pyriform lobe were found to be unreliable. Although we knew from pilot experiments that small electrolytic lesions in the pyriform region could be epileptogenic, and facilitate kindling (see also refs. 2, 3 and 16), we had found that large electrolytic lesions were less epileptogenic (possibly because the intact tissue was further removed from the site of ion deposits). Consequently, we selected this technique for producing large bilateral lesions in the pyriform lobe. The possibility of lesion-induced activity at distant sites remained, however, so we included those animals that had sustained sufficiently large radio frequency lesions. Three Teflon-coated stainless steel electrodes 240 ~zm in diameter, were implanted along the anterior/ posterior axis of the pyriform lobe in 20 animals. A bipolar stimulating electrode was also implanted into the lateral septal nucleus. At the time of surgery, a 2 mA anodal current was passed via the anterior electrodes, 30 s to one hemisphere followed by 30 s to the other. One week later, similar currents were passed via the middle 2 electrodes. The final 2 lesions were made one week later. It was necessary to produce these lesions in stages to ensure the survival of the animal. After a final one week recovery period, kindling was begun. Hippocampal lesions. An oval shaped opening was cut into the skull overlying the hippocampus in 8 animals. The overlying neocortex and the dorsal hippocampus were then removed by aspiration. A Tefloncoated stainless-steel bipolar stimulating electrode was implanted into the lateral septal nucleus. Two weeks following surgery, kindling was begun. Control animals and kindling procedure. Ten control animals were prepared with stimulating electrodes in the lateral septal nucleus. Kindling was begun 2-4 weeks following surgery. All animals were stimulated once per day with a 1-s train of biphasic,
rectangular pulses at 60 Hz. The current was initially set at 400/~A (800 ~A peak to peak). If this intensity did not produce an epileptiform afterdischarge (AD), then it was raised in 200-#A steps until an AD was produced. Similar intensity increases were made in randomly selected control animals. It was our intention to keep the current intensity well above threshold. We have found in previous experiments that kindling rates are highly variable when the stimulation intensity is near threshold levels (see also ref. 21). Stimulation was continued until the animal showed 2 consecutive generalized convulsions, consisting of a bilateral forelimb clonus, clonic rearing and a loss of postural control (falling). At the end of this, and all subsequent experiments, the brains were removed, fixed, sectioned and stained with thionin. Electrode placements and the extent of lesions (or knife-cuts) were then determined.
Experiment 2 - - the effect of dorsal hippocampal vs ventral hippocampal/entorhinal lesions on amygdala kindling The dorsal lesions were produced as in the previous experiments. Radio frequency lesions were attempted for the ventral lesions but were again found to be unreliable. Consequently, the ventral lesions were made electrolytically. In order to ensure that these lesions included ventral hippocampus and adjacent entorhinal cortex, the electrode was lowered, at an angle of 30° anterior to posterior, until the 1.0-mm bared tip was in the mid-caudal portion of the entorhinal cortex. A 2-mA current was then passed via the electrode for 30 s, after which the electrode was retracted dorsorostrally until the tip was presumed to be in the ventral hippocampus. The 2-mA current was reapplied. The electrode was moved to the other hemisphere and the procedure repeated. A bipolar stimulating electrode was then placed in the basolateral nucleus of the amygdala. A control group of 14 animals were prepared with a bipolar stimulating electrode in the amygdala. Two to 4 weeks following surgery, kindling was begun. The kindling procedure was as described above.
Experiment 3 - - the effect of knife-cuts in various amygdalar pathways Ninety-eight male hooded rats were used in this
266 experiment. Knife-cuts were made using the encannulated knife technique of Sclafani and Grossman 27. In this procedure, a cannula with an angled tip is lowered to a point above the intended site of transection. The wire is then pushed out of the cannula for several millimeters (depending upon the intended width of the cut). The cannula is lowered for the intended depth of the cut, after which the wire and the cannula are withdrawn. Knife cuts were made in all pathways except the stria terminalis, where we were unable to make reliable transections using this technique (the compact fiber bundle may have been deflected rather than cut by the curved wire knife). Consequently, we used electrolytic lesions to damage this pathway. Anterior coronal cuts. One group of 10 animals received unilateral knife cuts in a ventral coronal plane anterior to the amygdala. These cuts were intended to disrupt communication anteriorly with the pyriform cortex, as well as with medial structures (via amygdalar fibers that run anteriorly before turning mediaUy11). An additional group of 10 animals re-
ceived bilateral cuts in the same region. Medial cuts. Cuts were positioned medial to the amygdala, to disrupt ventral-amygdalofugal (VAF) fibers, in 20 animals. Ten of these received unilateral cuts, while the remaining 10 received bilateral cuts. Posterior coronal cuts. Cuts were made in a ventral coronal plane posterior to the amygdala in 20 animals (10 unilateral and 10 bilateral). These cuts were intended to disrupt communication with the posterior entorhinal cortex and the hippocampus 12. Stria terminalis lesions. Electrodes made of Tefloncoated 240/~m nicrome wire were used to pass 1 mA of anodal current in the region of the stria terminalis for 20 s. Ten animals received unilateral lesions and 10 animals received bilateral lesions. Control groups. The encannulated knife was lowered, without extending the knife, in a group of 18 control rats. Six groups of 3 control rats received the cannulae in the same placements as in the 6 knife cut groups. Kindling procedures. After the cuts were made, all groups received a bipolar nichrome wire electrode into both the amygdala and the dorsal hippocampus. The kindling stimulation was applied to the amygdala, and kindling procedures were as previously described. RESULTS
/
Fig. 1. For all experiments reported in this paper, electrode tips were found to be located within the regions indicated. (Atlas diagrams are from Paxinos and Watson19.)
Experiment 1 - - the effect of pyriform lobe vs hippocampal lesions on septal kindling The location of electrode tips for this and subsequent experiments is shown in Fig. 1. The extent of the lesions for this experiment is shown in Fig. 2. Two of the animals with hippocampal lesions died during the course of kindling. It was difficult to produce large bilateral lesions in the pyriform lobe, even in 3 stages, without killing the animals. Consequently, from the 20 animals that sustained lesions, we obtained only 10 survivors. Several of these had to be force fed during the first week after surgery. Feeding behavior eventually recovered, however, and the surviving animals, although lighter than controls, appeared active and healthy. As can be seen in Fig. 2, the resulting pyriform lobe lesions were not complete but the total damage was comparable to that of the hippocampal group. The electrode of one of the control animals was outside of the amygdala, so this ani-
267
PYRIFORM LOBE LESION SMALLEST
LARGEST
1.7MM
"0.8MM
-3.3MM
DORSAL HPC LESION SMALLEST
LARGEST
-3.3MN
"4.3MM
-5.3MM
Fig. 2. The smallest and largest pyriform lobe lesions as well as the smallest and largest hippocampal lesions are shown above. Note that the pyriform lobe lesions are represented by cross-section through the c e n t e r s of the 3 anterior/posterior lesions placed in those lobes. There was considerable reduction in volume of damage half way between lesion sites (about 70-100% reduction for the small lesions and a 30-60% reduction for the large lesions).
268 mal's data were excluded from the analysis. The data and analyses are presented in Table I. As can be seen, the group with the pyriform lesion required a significantly greater number of stimulations to complete kindling than either the control group or the group with the hippocampal lesions. The mean kindling rate was actually somewhat faster in the group with hippocampal lesions, but the differences did not reach significance. None of the measures of A D strength (duration, spike amplitude, etc.) were significantly different between the groups, although the A D durations in the group with pyriform lobe lesions were somewhat shorter than controls upon completion of kindling (P = 0.1). None of the animals with pyriform lesions required increases in the intensity of stimulation in order to evoke ADs. Three of the animals with hippocampal lesions required a 100% increase in stimulation intensity. Experiment 2 - - the effects o f dorsal hippocampal vs ventral hippocampal/entorhinal lesions on amygdala kindling The extent of the dorsal and ventral lesions are shown in Fig. 3. Two animals with dorsal lesions were lost during the course of kindling (they had received only 2 and 4 stimulations, respectively). The remaining animals were healthy and active. Table II shows the data and analyses. There were no significant differences between the groups in the number of stimuTABLE I Septal kindling Number of ADs to first stage 5 convulsion. Lesion Amygdala/pyriform (n=lO)
Hippocampus (n=6)
Control (n = 9)
41.6 (26-59)
23.5 (10-45)
30.3 (13-49)
Experiment 3 - - the effects o f knife cuts in various amygdalar pathways Minimal, maximal and/or representative cuts (or lesions) for each structure are shown in Fig. 4. Data and analyses are shown in Table III. There were no significant differences between the effects of unilateral and bilateral cuts, so the groups were pooled for pairwise comparisons. Stria terminalis lesions produced a significant facilitation of kindling. Cuts in the coronal plane between the amygdala and the ventral hippocampus also resulted in a significant facilitation of kindling. Cuts in the coronal plane anterior to the amygdala retarded kindling, but the effect was not very large. Although there were no significant differences in A D durations at any time during the experiment, there appeared to be a tendency towards longer first trial A D durations in the groups with stria terminalis lesions or posterior coronal cuts. Propagation to the hippocampus, however, was strikingly affected in the groups with posterior ventral coronal cuts. These animals showed no sign of propagated activity to the hippocampus during early stages of kindling and little or no propagation even after generalized convulsions had developed. If propagated activity was measured in terms of either absolute spike amplitude or amplitude relative to background E E G amplitude (signal to noise) there was no overlap between the posterior ventral coronal cut groups and any other group tested. Examples of these discharges are shown in Fig. 5. TABLE II
ANOVA Between Within Total
lations required to reach a stage 5 convulsion. There were also no significant differences in any of the measures of the A D , except that all animals with ventral hippocampal lesions required a 100-200% increase in stimulation intensity in order to trigger ADs.
Amygdala kindling Number of ADs to first stage 5 convulsion.
ss df 1344.1 2 3015.9 22 4360.0 24
Pairwise Pyriform vs control t = 2.25, P = 0.036
MS 672.1 137.1
F 4.9
Pyr vs hpc t = 3.02, P = 0.009
P 0.017
Control vs hpc t = 1.01, N.S.
Lesion Dorsal hippoeampus n=6
Ventralhippocampal
Control
.13.3 (8-27) ANOVA N.S.
12.4 (10-18) N.S.
12.1 (8-23) N.S.
269
DORSAL HPC LESION SMALLEST
LARGEST
-3,
-4
-5,
VENTRAL HPC LESION SMALLEST
LARGEST
Fig. 3. The smallest and largest lesions of the dorsal hippocampus and ventral hippocampus/entorhinal cortex are shown above.
270 A further difference between the groups with either posterior ventral coronal cuts or stria terminalis lesions and the other groups was that post-ictal spiking appeared
earlier in the
former
groups
clear that the hippocampus is not critical for either septal Or amygdala kindling. Large dorsal hippocampal lesions affected neither septal nor amygdala kindling. Large ventral hippocampal/entorhinal le-
DISCUSSION
sions had no effect on amygdala kindling. Although epileptogenic effects of the D C lesions on intact pyriform lobe tissue might have masked an otherwise de-
There are several conclusions that can be drawn on the basis of these lesion and knife-cut data. First, it is
pressant effect of the lesions, Expt. 3 showed that a blockade of c o m m u n i c a t i o n between the amygdala and the hippocampus (confirmed by recording hippo-
(Fig. 5).
A
B
C
STRIA LESION
.
V A F KNIFE CUT
A
B
A
B
.0
POSTERIOR CORONAL CUT
ANTERIOR CORONAL CUT
Fig. 4. The extent of the lesions or knife cuts are illustrated above. The smallest (A), most representative (B) and largest (C) lesions of the stria terminalis are shown in the top row. The number in the upper left hand corner indicates the AP coordinate with reference to bregma (Pellegrino and Cushman2°). A typical ventral-amygdalofugal (VAF) cut is shown in the second row. The extent of the cut is indicated by the vertical line in each section. There was little variation in these cuts and nearly all reached the base of the brain between AP coordinates +2.0 and -0.4 ram. The posterior and anterior coronal cuts are illustrated in the bottom row. The largest (A) and smallest (B) cuts, as reconstructed from sagital sections, are shown for both types of cuts.
271 TABLE III Number of afterdischarges (ADs) required to kindle to generalized seizure Unilateral cuts
Bilateral cuts
Stria terminalis
r X
(5-12) 7.8 n=9
(4-9) 6.4 n=8
Posterior coronal
r X
(4-12) 7.9 n=9
(5-11) 7.8 n=9
VAF
r X
(6-14) 10.8 n = 10
(7-31) 12.9 n = 10
Anterior coronal
r X
(10-21) 13.7 n=10
(9-19) 14.2 n=9
Control
r X
(8-13) 10.8 n=8
(8-15) 11.3 n=8
ANOVA Source Total Placement Uni- or bilateral Placement unior bilateral Error
SS 11326 593.7
df 89 4
ms 148.4
2.3
1
2.3
29.5 887
4 80
7.4 11.1
Pairwise (Tukey): Stria vs control Posterior vs control VAF vs control Anterior vs control
F 13.4
-
P 0.001
0.21
N.S.
0.66
N.S. -
P < 0.01 P < 0.05 N.S. P < 0.09
campal activity) was actually a c c o m p a n i e d by a facilitation of kindling. These results are different from those of Savage et al. 26 who r e p o r t e d that knife cuts in the entorhinal cortex r e t a r d e d amygdala kindling, and of Dashieff and M c N a m a r a 4 who r e p o r t e d that colchicine application to the d e n t a t e gyrus r e t a r d e d entorhinal cortex kindling. T h e r e are several possible explanations for these differences. Most of the experiments that have tested the effects of lesions have used kindling stimulations that were below or just above threshold intensity (for A D ) . Pinel et al. 21 have shown that kindling rates can be quite variable near threshold, and are usually prolonged. This appears to be due to the fact that n e a r threshold stimulation often produces weak discharges that contribute little to the kindling process 21 (Racine, unpub-
lished observations). Even small alterations in A D threshold might account for an a p p a r e n t increase in kindling rates when the stimulation intensity is held near the threshold levels. W e have used stimulation levels that were clearly suprathreshold to ensure that we were measuring kindling rates and not a secondary effect of threshold change. The colchicine results of Dashieff and M c N a m a r a 4 could be due to an effect of the drug outside of the site of application. The authors state that they had to exclude 9 animals that showed clear entorhinal cortex damage. M a n y of their remaining animals showed substantial increases in threshold and reduction of A D durations. Nevertheless, they still required fewer total seconds of A D (though an increased n u m b e r of A D s ) to complete kindling. The authors conceded that the colchicine may have had some effect outside the injected area. Also, Sutula et al. 28 found no effect of colchicine, administered to the dentate, on entorhinal cortex kindling (although they did find an increase in A D threshold). T h e r e is certainly no question, from our results, that the h i p p o c a m p u s is n o t a critical link in the d e v e l o p m e n t and p r o p a g a t i o n of kindled seizures, and its activation m a y even slow down the development of kindling. This interpretation is consistent with the fact that the h i p p o c a m p u s is one of the slowest kindling sites in the brain 1°'22'24. In fact, when convulsions do finally a p p e a r during h i p p o c a m p a l kindling, they often occur during a p e r i o d of s u p p r e s s e d discharge in the h i p p o c a m p u s 22. This p e r i o d of suppression follows the h i p p o c a m p a l A D , and the epileptiform activity of other limbic sites is often of largest amplitude during this period. A l t h o u g h the ventral h i p p o c a m p a l / e n t o r h i n a l lesions did p r o d u c e large increases in A D thresholds, this m a y have been due to an e n c r o a c h m e n t of the lesion into the amygdala (near the vicinity of the stimulating electrode). W e had earlier a t t e m p t e d to test amygdala kindling in animals with pyriform lobe lesions anterior to the electrode. W e could not even elicit A D s from these animals, p r e s u m a b l y for the same reason. A n o t h e r conclusion that can be drawn is that the pyriform lobe region appears to be facilitatory for kindling. Medium-sized bilateral lesions of the pyriform lobe significantly r e t a r d e d septal kindling, and ventral coronal knife-cuts b e t w e e n the a m y g d a l a and the anterior pyriform cortex r e t a r d e d a m y g d a l a
272 ANTERIOR VENTRAL CORONAL CUT S No. 4
v
16
:b,W,~vb.:::-:L.W~t.:.. . . . . . .
POSTERIOR VENTRAL CORONAL CUT S No. $ I
]
7
:'/q~ ;
II~.~V
......
Fig. 5. The development of the afterdischarge (AD) during kindling is shown for 2 representative animals. The top 4 traces were taken from an animal with an anterior ventral coronal cut. The 1st, 5th and 16th ADs are shown. The last AD was accompanied by a fully generalized convulsion. The development in this animal was typical for that group and for every other group except those with posterior ventral coronal cuts. Traces from a typical animal from the latter group are shown in the bottom 4 traces. The 1st, 3rd and 7th ADs are shown for this animal. The 7th AD was accompanied by a fully generalized convulsion. Note that the propagation to the hippocampus (HPC) is strong from day 1 in the animal with the anterior cut and never develops for the animal with the posterior cut. Also, note the post-ictal spikes that appear near the end of the last trace in the animal with the posterior cuts. These post-ictal spikes developed within the first few stimulations in the posterior cut animals, but they required at least 2 or 3 additional ADs for animals in the other groups.
kindling. The results of both the hippocampal lesions and the pyriform lobe lesions are consistent with those presented in the previous paper in which we reported that inter-ictal spikes appear to develop more readily in the pyriform region than in the hippocampus 23. As mentioned in the Methods section, we had initially attempted to produce these lesions with the radio frequency technique. Although this technique proved unreliable in our hands, we did eventually obtain 3 animals with lesions as large as those produced here. Those animals also required a greater than normal number of stimulations to kindle. These results are consistent with those of Le Gal La Salle 13who re-
ported that amygdala lesions retarded kindling from the bed nucleus of the stria terminalis and of Cain 1 who found that amygdala lesions retarded olfactory bulb kindling. The stria terminalis lesions facilitated amygdala kindling. This finding confirms that reported by Engel and Katzman 7 who also found a significant facilitatory effect of stria terminalis lesions. They suggested that this effect was due to a disruption of ascending noradrenergic fibers that run through the stria terminalis on their way to the amygdala 14,29. Another possibility is that the amygdalar projections, via the stria terminalis, to diencephalic structures are predominantly inhibitorys,6,25. Disruption of these fibers
273 might then be expected to facilitate the relay of epileptogenic activity into diencephalic and brainstem structures over remaining pathways. These alternative possibilities also apply to the facilitation pro-
the nucleus accumbens, ventral pallidum and substantia i n n o m i n a t a may be important in the propagation of the discharge from pyriform lobe sites (see
duced by posterior ventral coronal cuts. Either of
also ref. 18). We have attempted several times to place lesions in these structures, but the survival rate
these pathways could be inhibitory in either direction resulting in a disinhibition of the primary (stimu-
has been near zero. A final conclusion to be drawn from these results is
lated) site or of one or more secondary sites to which the primary site projects. The ventral amygdalofugal cuts had no significant
that it is very difficult to block kindling. None of the lesions or knife-cuts in these experiments did so. Even the pyriform lobe lesions only produced a rela-
effect. This was surprising for us because we had
tively mild suppression of kindling. This provides ad-
found what appeared to be a retarding effect in a pilot experiment prior to beginning this study. Those
ditional support for our argument in the previous paper 23 that m a n y sites are capable of developing a
cuts were more anterior to the 'corrected' placements used here. If the pilot results reflected a genuine effect, there is a possibility that the region of
chronic epileptogenic responsivity, with the pyriform
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